The major focus of this proposal is to define the Mediator complex as a therapeutic target in AML. Cancer cells are often dependent on aberrant transcriptional programs to maintain their tumorigenic state. Therefore, rationale exists to therapeutically target oncogenic transcription factors. A major obstacle in this pursuit arises fro the structure of DNA-binding transcription factors, which generally lack the deep hydrophobic pockets that are amenable to small-molecule-based inhibition. As an alternate strategy, my PhD advisor's laboratory has undertaken efforts to target transcriptional coactivators, which have already revealed promising opportunities for drug discovery. My research proposal will focus specifically on the Mediator complex, a critical co-activator that relays regulatory signals from sequence-specific transcription factors to the core transcriptional machinery. The central hypothesis of my proposal is that targeting individual subunits of Mediator will provide a means of interrupting the function of select oncogenic transcription factors. I began evaluating this hypothesis by employing a functional-genetic approach in which I systematically knocked down all 33 Mediator subunits and measured the impact on cell viability in non-transformed and leukemia cell lines. For this purpose, I have focused on a type of the hematopoietic cancer acute myeloid leukemia (AML) that is initiated by the MLL-AF9 oncoprotein. My screening strategy has pinpointed the Med12 subunit of Mediator as being selectively required for leukemia proliferation, whereas other subunits are more generally required for cell proliferation. My proposal will focus on evaluating the mechanism of addiction of AML to Med12. This will entail cellular, transcriptional, and epigenomic characterization of Mediator function in AML, as well as an in vivo exploration of Med12 function. The studies I propose here will establish a co-activator complex as a novel target through which leukemogenic transcriptional programs can be drugged.